Simultaneous and Visual Detection of KPC and NDM Carbapenemase-Encoding Genes Using Asymmetric PCR and Multiplex Lateral Flow Strip

Carbapenem-resistant Enterobacteriaceae (CRE) infections constitute a threat to public health, and KPC and NDM are the major carbapenemases of concern. Rapid diagnostic tests are highly desirable in point-of-care (POC) and emergency laboratories with limited resources. Here, we developed a multiplex lateral flow assay based on asymmetric PCR and barcode capture probes for the simultaneous detection of KPC-2 and NDM-1. Biotinylated barcode capture probes corresponding to the KPC-2 and NDM-1 genes were designed and cast onto two different sensing zones of a nitrocellulose membrane after reacting with streptavidin to prepare a multiplex lateral flow strip. Streptavidin-coated gold nanoparticles (SA-AuNPs) were used as signal reporters. In response to the target carbapenemase genes, biotin-labelled ssDNA libraries were produced by asymmetric PCR, which bond to SA-AuNPs via biotin and hybridise with the barcode capture probe via a complementary sequence, thereby bridging SA-AuNPs and the barcode capture probe to form visible red lines on the detection zones. The signal intensities were proportional to the number of resistance genes tested. The strip sensor showed detection limits of 0.03 pM for the KPC-2 and 0.07 pM for NDM-1 genes, respectively, and could accurately distinguish between KPC-2 and NDM-1 genes in CRE strains. For the genotyping of clinical isolates, our strip exhibited excellent consistency with real-time fluorescent quantitative PCR and gene sequencing. Given its simplicity, cost-effectiveness, and rapid analysis accomplished by the naked eye, the multiplex strip is promising auxiliary diagnostic tool for KPC-2 and NDM-1 producers in routine clinical laboratories.


Introduction
Te number of infections caused by carbapenem-resistant Enterobacteriaceae (CRE) strains has increased in recent years [1,2]. Carbapenemases are primarily classifed into Ambler classes A, B, and D [3,4]. Among CRE species, Klebsiella pneumoniae carbapenemase (KPC) and New Delhi-metallo-β-lactamase (NDM) pose a major threat, being the most prevalent in Klebsiella pneumoniae and Escherichia coli, respectively, in China [5] and causing an alarming increase in infection rates over the last year [6]. Infectious diseases caused by CRE have become an urgent concern for healthcare institutions because they are associated with high mortality and morbidity rates [7,8]. Moreover, susceptible bacteria readily become resistant to carbapenems through the transfer of plasmids between Enterobacteriaceae [9,10]. Tus, there is an urgent need for the rapid diagnosis of CRE infections.
Carbapenemase detection, which involves phenotypic screening and genotype detection, is the primary method used for the clinical diagnosis of CRE infections. Phenotypic tests include the Carba NP test [11], carbapenem inactivation method [12], modifed Hodge test [13], carbapenemase inhibitor enhancement test [14], colour medium screening [15], and fight mass spectrometry [16]. Tese phenotypic screening methods show good specifcity and low cost but often sufer from low sensitivity and slow turnaround times [17,18]. Genotype determination mainly involves fuorescence quantitative PCR [19], enzyme immunochromatography technology [20][21][22][23], and genome sequencing [24]. Antibody-based immunochromatographic methods are fast and easy to operate but require expensive antibodies. Genotyping methods signifcantly increase the detection efciency with high accuracy and sensitivity [25,26]; however, the requirement for expensive instruments and strict experimental conditions limit their application in on-site detection and in remote areas.
Recently, lateral fow test (LFT) technology was applied in rapid nucleic acid detection [27][28][29]. Given its simplicity, rapid turnaround, low cost, and user-friendliness, LFT has become one of the most widely used point-of-care testing (POCT) methods for rapid diagnostic systems [30,31]. Notably, LFT is further exploited for the simultaneous detection of multiple analytes from a single sample, commonly known as multiplexed detection, which has attracted considerable attention [21,22,32]. Multiplex testing reduces sample volume, analysis time, and cost. Owing to these advantages, LFT is widely used to detect drugs of abuse [33], nucleic acids [34], proteins [35], and bacteria [36][37][38].
In this study, we propose a multiplex assay for detecting KPC and NDM genes using an asymmetric PCR technique and a barcode-capture probe-based lateral fow test strip. An asymmetric PCR-based technique was employed to obtain adequate amounts of biotinylated single-stranded DNA products of KPC and NDM (biotin-ssDNA). Biotin-ssDNA reacts with streptavidin-coated gold nanoparticles (SA-AuNPs) on the conjugate pad to form a AuNP-SA-biotin-ssDNA (AuNP-ssDNA) complex. AuNP-ssDNA is hybridised to barcode capture probes when the sample fows through the test lines; thereby, AuNPs are immobilised on the test zones of the strip sensor to produce a red line. Te visual signals could be read by the naked eye and quantifed with a GIC-H1 portable analyser. Te multiplex strip for typing KPC-2 and NDM-1 genes has great potential for application in basic microbiology laboratories.  Figure S2), respectively, and the sequences of the DNA fragments are shown in Table S2 of Supplementary Material. For KPC-2 and NDM-1 gene testing, the two plasmids (pUC57/kpc and pUC57/ndm) were artifcially synthesised and not isolated from bacteria, comprising partial KPC-2 and NDM-1 sequences (Tsingke Biotechnology Co., Ltd., Beijing, China). Te synthesised plasmid did not contain any other beta-lactamase genes. Te concentrations of the pUC57/kpc and pUC57/ndm plasmids were frst determined by NanoDrop spectrophotometry.

Strain
Culture and DNA Extraction. Bacterial strains were cultured on Columbia blood agar plates, and total DNA was extracted using the MagIso DNA/RNA isolation kit according to the manufacturer's instructions. Te extracts were stored at −20°C before use.

Preparation and Modifcation of Gold Nanoparticles.
All glassware used in the preparation was thoroughly cleaned in aqua regia (three parts HCl and one part HNO 3 ), rinsed in double-distilled water, and oven-dried prior to use. Gold nanoparticles (AuNPs) were prepared using the citrate reduction method. Briefy, 100 mL of 0.01% HAuCl 4 in double-distilled water was boiled and mixed with 4 mL 1% trisodium citrate with vigorous stirring. When the colour turned wine red, the heating source was withdrawn, and the colloid solution was stirred for another 15 min. Te colloidal gold solution was photographed using TEM. Prior to modifcation, the pH of the AuNP solution was adjusted to 8.0 by addition of 0.1 mol/L K 2 CO 3 . Next, 1 mL AuNP solution was mixed with 20 μL streptavidin solution (0.1 mg/ mL). Te mixture was shaken for 1 h, followed by addition of 0.1% BSA as a blocker. Te mixture was incubated for 30 min at room temperature. Te solution was centrifuged to remove unconjugated streptavidin at 10000 rpm for 20 min at 4°C. Te pellet was reconstituted in 20 mL of the proper storage bufer (1% PBS, 1% BSA, 0.25% sucrose, 1% Tween-20, and 0.05% NaN 3 ). Te SA-AuNPs were fnally stored at 4°C for further use. Te absorption spectra of the AuNP and SA-AuNP solutions were scanned at λ 300-750 nm with a UV spectrophotometer.

Preparation of Streptavidin-Biotin-DNA Probe
Conjugates. Te capture probes on test lines 1 and 2 (T1 and T2) were designed to immobilise the AuNP conjugates corresponding to KPC-2 and NDM-1, respectively. Tree types of capture probe (Cp) were prepared: streptavidinbiotinylated Cp1 conjugates for T1 (KPC-2), streptavidinbiotinylated Cp2 conjugates for T2 (NDM-1), and streptavidin-biotinylated Cp3 conjugates for the control line. Streptavidin was dissolved in 10 mM (phosphatebufered saline (PBS), pH 7.4) to a fnal concentration of 0.1 mg/mL. Next, 8 μL streptavidin (0.1 mg/mL) and 1 μL Cp (50 μM) with a ratio of 8 : 1 were incubated at room temperature for 1 h to provide stable binding. Te sample solutions were fltered (30 kDa, Amicon Ultra, Merck Millipore, Burlington, MA, USA) for 30 min at 6000 rpm to remove free Cp. Conjugated streptavidin-biotin-Cp, which remained in the flter owing to the large molecular size of streptavidin, was removed from the flter membrane and resuspended. Streptavidin-biotinylated-Cp conjugates were dispensed onto three regions of a nitrocellulose membrane to prepare two test lines and a control line.

Detection of KPC and NDM
Genes. DNA extracts from diferent strains, which contained with or without KPC and NDM gene, were subjected to the established asymmetric PCR system. Two types of plasmid DNA containing the KPC-2 or NDM-1 genes, used as standards, were tested. ddH 2 O was used as a blank control. Next, 10 μL asymmetric PCR amplifcation products and 90 μL 0.01 mol/L PBS diluent were mixed in an Eppendorf tube. Finally, the lateral fow strip was placed in the mixture for 15 min. Te corresponding intensities were measured using the GIC-H1 portable analyser that scanned the test and control line and read the intensity of the refected light.

Carbapenemase Detection for Clinical Strains.
To validate our method, we used 23 clinical isolates of Enterobacteriaceae to evaluate the KPC and NDM genes; these strains were either positive or negative for KPC or NDM. We used our strip and sequencing methods, and we compared the results between the methods. A meropenem disk with phenylboronic acid (PBA) and EDTA has also been used to detect double carbapenemase producers (serine and metalloβ-lactamase, respectively) [11,13].

Principle of the Multiplex Strip for Sensing KPC and NDM
Genes. Te working principle of the nucleic acid lateral-fow test strip is shown in Figure 1. In this study, an integrated multiplex assay based on asymmetric PCR techniques and barcode lateral fow tests was used to detect genes in CRE strains. First, primers for KPC-2 and NDM-1 genes were designed with reference to the GenBank data on KPC and NDM genes. To react with streptavidin-coated AuNPs, the reverse primers of the KPC-2 and NDM-1 genes were tailored with a biotin molecule at their 5 ′ end. Upon amplifcation of the target carbapenem-resistant gene by asymmetric PCR, a library of asymmetric PCR products (biotin-ssDNA) was produced. Biotin-ssDNA1 was generated in the presence of KPC-2, and biotin-ssDNA2 corresponded to the NDM-1 gene. With co-occurrence of KPC-2 and NDM-1, the asymmetric PCR products consisted of biotin-ssDNA1 and biotin-ssDNA2 (Figure 1(a)). Tree types of biotin-DNA barcode Cps were designed: biotin-Cp1 complementary to the partial sequence of biotin-ssDNA1 (KPC-2), biotin-Cp2 complementary to the partial sequence of biotin-ssDNA2 (NDM-1), and biotin-Cp3 complementary to the reverse primer sequences of KPC-2 and NDM-1. Tese biotin-capture probes reacted with streptavidin to form a streptavidin-biotin-Cp complex (barcodes 1, 2, and 3), which was dispensed onto diferent zones of the nitrocellulose membrane as capture probes for the test and control lines; T1 and T2 corresponded to KPC-2 and NDM-1, respectively. AuNP-SA conjugates were employed as reporters and loaded onto the conjugate pad. Visual detection of the sample reaction solution was performed using a strip-sensing system consisting of four components: a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorption pad (Figure 1(b)). Te presence of KPC-2 and/or NDM-1 resulted in the generation of biotin-ssDNA products. A sample solution containing the biotin-ssDNA products was then applied to the sample pad. Te solution was migrated by capillary action, passed through the conjugate pad, and rehydrated the AuNP-SA conjugates. Biotin-ssDNA reacts with AuNP-SA to form AuNP-SA-biotin-ssDNA complexes (AuNPs-SA-biotin-ssDNA) which continue to migrate along the strip. Because the ssDNA products were complementary to the barcode on the immobilised test lines, the above complexes were captured in the test zones by hybridisation between ssDNA and barcode probes. Te accumulation of AuNPs in test zones 1 and 2 is visualised as characteristic red bands. Excess biotin-labelled reverse primers reacted with AuNPs-SA conjugates to form AuNPs-SA-biotin-R primers, which continued to migrate and were captured in the control zone by hybridisation with barcode 3, thus generating a third red band (Figure 1(c)). In the absence of KPC-2 and NDM-1, no red band was observed in the test zone owing to the failure of ssDNA production. Te biotin-modifed reverse primers of the KPC-2 and NDM-1 genes were excess, and a red band was always formed in the control zone, confrming the proper functioning of the strip biosensor (Figure 1(d)). Figure 1(e) shows the interpretation of reagents and probes used in the strip and asymmetrical PCR system. Te positive results of KPC-2, NDM-1, both KPC-2 and NDM-1, and invalid test result are explained in Figure 1(f ).

Characterization of the Prepared and Modifed Gold
Nanoparticles. Te colloidal gold solution prepared by citrate reduction was clear red (Figure 2(a)). Te gold nanoparticles were dispersed with a diameter of 30-50 nM, as confrmed by transmission electron microscopy (TEM,   Figure 2(b)). A typical absorption peak at λ 520 nm was observed in the colloidal gold solution using the UV spectrophotometer (tube a and spectrum a, Figure 2(c)). Tese fndings indicated that colloidal gold was successfully prepared. After modifcation, the streptavidin-coated colloidal gold solution remained red (tube c), and the corresponding absorption peak at 520 nm remained unchanged (spectra c). Upon addition of 10% sodium chloride to the colloidal gold solution, the colour changed from burgundy to grey (tube b). However, adding sodium chloride had an efect on the colour change (tube d), and absorbance peaks at λ 520 nm were detected by using the UV spectrophotometer. Tis occurred because outer streptavidin prevented the aggregation of colloidal gold in a high-salt solution, suggesting successful modifcation of colloidal gold with streptavidin.

Feasibility of the Multiplex Strip for Sensing KPC-2 and NDM-1.
We investigated the feasibility of our method by using two types of plasmids containing KPC-2 (pUC57/kpc) and NDM-1 (pUC57/ndm) sequences (100 pM) as targets. Te presence of both the KPC-2 and NDM-1 genes resulted in two red test lines (Figure 3), indicating that the biotin-ssDNA libraries successfully connected AuNPs-SA onto the barcode probes of KPC-2 and NDM-1. Te presence of KPC-2 or NDM-1 led to a single red test line in the test zone. Tese results suggest that only one type of biotin-ssDNA was generated in response to KPC-2 or NDM-1, which immobilised AuNPs-SA on the test line. Only the control line appeared in the absence of KPC-2 and NDM-1. Terefore, the established method has a robust ability to accurately and rapidly detect carbapenemase genotypes.

Optimization of the Asymmetric PCR Annealing
Temperature. Annealing temperature is an important experimental parameter afecting the analytical performance of asymmetric PCR. Te maximum production of the target biotin-ssDNA and minimum production of by-products were achieved at 55°C for the KPC-2 gene at 10 pM (Figure 4(a)). Meanwhile, the test line of KPC-2 showed the highest signal (585.33, Figures 4(c) and 4(d)). For the NDM-1 gene at 10 pM, the amount of biotin-ssDNA product for NDM-1 at 55°C was marginally higher than that at other temperatures (Figure 4(b)). With the biotin-ssDNA products on the strip, a relatively stronger signal of 435.33 was obtained at 55°C (Figures 4(c) and 4(d)). Terefore, 55°C was selected as the best annealing temperature for amplifying KPC-2 and NDM-1.

Optimization of the Ratio of the Forward to Reverse
Primer. Te ratio of forward to reverse primers (F/R ratio) is an important factor of biotin-ssDNA production. In the preparation of target biotin-ssDNA, the limited forward primers would be exhausted and the excess reverse primers would amplify the dsDNA in the early phase of asymmetric PCR to produce numerous ssDNA amplicons. At 10 pM of the target gene of KPC-2 and NDM-1, we observed a small diference between the gel electrophoresis bands of asymmetric PCR at diferent primer ratios (Figures 5(a) and 5(b)). However, the strip signals in Figure 5(c) appear diferent in staining, and the corresponding signal intensity in Figure 5(d) shows that the 1 : 20 F/R ratio was optimized based on the high signal intensity of KPC-2 and NDM-1 (544.67 and 515.33, respectively). Terefore, an F/R ratio of 1 : 20 was selected for subsequent assays.

Optimization of the Cycle Number.
In asymmetric PCR, the cycle number is a vital factor afecting the production of biotin-ssDNA and thus needed to be optimized.
With an increase in the cycle number, the production of biotin-ssDNA for KPC-2 (10 pM) increased and tended to peak at 35 cycles (Figure 6(a)). Te strip achieved its highest intensity (533.33 ) after 35 cycles (Figures 6(c) and 6(d)). Agarose gel electrophoresis of the DNA bands showed that biotin-ssDNA products for NDM-1 (10 pM) increased as the number of cycles increased from 25 to 40 and decreased thereafter ( Figure 6(b)). For the strip, the maximum signal (505.33 ) for NDM-1 occurred at 40 cycles (Figures 6(c) and 6(d)), which is consistent with the agarose gel electrophoresis analysis. Considering the compatibility of the asymmetric PCR system for KPC-2 and NDM-1, the best assay conditions were selected at 40 cycles.

Sensitivity Analysis.
To investigate the sensitivity of the strip sensor and establish standard curves for KPC-2 and NDM-1, the concentrations of the pUC57/kpc and pUC57/ ndm plasmids were set in 10-fold dilution. Under optimized experimental conditions, pUC57/kpc and pUC57/ ndm were added to the asymmetric PCR system, and the obtained biotin-ssDNA products were applied to the test strip. Te strip signals were enhanced with increasing concentrations of the target KPC-2 and NDM-1, and the strip intensities were proportional to the logarithm of the KPC-2 and NDM-1 concentrations (from 0.1 to 100 pM, Figures 7(a) and 7(b)). Te resulting calibration curves were I � 2746.32 + 201.82log 10 C for KPC-2 and I � 2700.61 + 199.03log 10 C for NDM-1 (inset in Figures 7(a)  and 7(b)). According to the principle of blank plus three times of standard, the detection limits were down to 0.03 pM for KPC-2 and 0.07 pM for NDM-1. Te red bands on the test zone were clearly visible even at 0.1 pM of KPC-2 and NDM-1, suggesting that the multiplex strip can be used for simultaneous visual detection of KPC and NDM genes without any specialized instrumentation. Terefore, this multiplex strip shows great promise for POCT detection of KPC-2 and NDM-1-the main markers of CRE.
3.6. Specifcity Analysis. Specifcity of the strip sensor was evaluated by adding a set of bacterial strains to the asymmetric PCR system. Two clinical strains carrying carbapenemase and eight standard strains carrying no carbapenemase were selected to test assay specifcity Journal of Analytical Methods in Chemistry (Table 1). Te asymmetric PCR products were sampled on lateral fow strips, and the results were observed after 10 min. Te results indicated the presence of KPC-2 in carbapenem-resistant K. pneumoniae and NDM-1 in carbapenem-resistant E. coli, while the eight standard strains including K. pneumoniae and E. coli showed negative results, similar to those for the blank control (ddH 2 O, Figure 8(a)). Fluorescence PCR, a sensitive method for the detection of carbapenemase genes [39], was performed on the aforementioned strains , which showed that the genotyping of the KPC and NDM genes was 100% consistent with the multiplex strip (Figures 8(d) and 8(e)). In addition, the phenotypes of the KPC and NDM enzymes were analysed by assessing the drug susceptibility of two carbapenem-resistant strains isolated from clinical samples. Imipenem-resistant K. pneumoniae and Escherichia coli became sensitive after the addition of boric acid and EDTA solution, respectively (Figure 8(b)). Te changes in susceptibility suggest that K. pneumoniae produced the KPC enzyme, the activity of which was inhibited by boric acid, and E. coli produced the NDM enzyme, the activity of which was blocked by EDTA. Te lateral fow test for typing carbapenemases further confrmed the existence of the KPC enzyme in K. pneumoniae and the NDM enzyme in E. coli (Figure 8(c)). For the two clinical CRE strains, the phenotypes of the KPC and NDM enzymes were consistent with the genotype test using fuorescent PCR and our strip test. In summary, this multiplex strip could accurately detect CRE strains containing KPC and NDM genes and had no nonspecifc reaction with other standard strains used in the assay.

Validation of the Strip for Testing KPC and NDM Genes in Clinical Samples.
Of the 23 clinical samples tested, we detected 12 K. pneumoniae with KPC-positive genes, 1 E. cloacae with KPC-positive genes, 2 K. pneumoniae with NDM-positive genes, 2 E. coli with NDM-positive genes, 1 E. oxytoca with NDMpositive genes, and 5 Enterobacter without carbapenemaseresistant genes (Table 1). Te identifcation of carbapenemase genes using the multiplex strip ( Figure S3) was consistent with the sequencing results (Tables S3 and S4). We performed phenotypic tests of bacterial antimicrobial resistance and found consistent results with those of the strip and sequencing analysis.     NDM, sensitivity: 100%). Te sensing probe used in the assay was also not cross-reactive (specifcity: 100%). Te results confrm that the strip sensor shows great potential in the typing of CRE genes in clinical samples.

Conclusion
Te multiplex lateral fow strip was developed for the simultaneous and visual detection of KPC and NDM genes in CRE strains using asymmetric PCR, barcoded capture probes, and streptavidin-coated gold nanoparticles. Asymmetric PCR enabled the production of numerous biotin-ssDNAs with high sensitivity. DNA barcodes designed as capture probes on the test and control lines enabled multiplex detection of carbapenemase genes in a one-pot assay system. Tis method has a shorter assay time and higher specifcity than traditional antimicrobial susceptibility testing methods. Compared with other available lateral fow immunoassays [40], our multiplex strip successfully detected the two major carbapenemases in CRE strains. Nucleic acid probe-based methods have the advantages of easy preparation, higher sensitivity owing to the integration of the amplifcation strategy, and lower costs than antibody-dependent lateral fow immunochromatographic assays [20][21][22]. Another advantage of this method is that the test strips can be stored at room temperature for longer periods [3]. Although the strip is convenient and user-friendly, it is limited by its reliance on ordinary PCR instruments and capacity for only detecting KPC-2 and NDM-1. Further work is required to adopt the isothermal amplifcation method and design a set of capture probes for sensing a greater variety of carbapenemase genes, including KPC, IMP, VIM, NDM, and OXA-48. Overall, the developed lateral fow assay exhibits high sensitivity and specifcity, providing a promising method for POCT in limitedresource settings, and ultimately prevents CRE transmission and outbreaks in hospitals.

Data Availability
Te data used to support the fndings of this study are available in the supplementary material of this article. Table S1 using Carba-5 strip. Figure S2 included information of plasmid pUC57 carrying blaKPC gene or blaNDM gene. Figure S3 included results of KPC-2 and NDM-1 testing on 23 Enterobacteriaceae samples using our strip. (Supplementary Materials)